1. Field of the Invention
The present invention is directed to a method of synthesizing sulfurized oligonucleotide analogs by reacting an oligonucleotide analog containing a trivalent phosphorous linkage with a dithiocarbonic acid diester polysulfide.
2. Discussion of the Background
It is well-known that most of the bodily states in mammals, including most disease states, are effected by proteins. By acting directly or through their enzymatic functions, proteins contribute in major proportion to many diseases in animals and man.
Classical therapeutics has generally focused on interactions with such proteins in an effort to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to directly inhibit the production of proteins involved in disease by interacting with the messenger RNA (mRNA) molecules that direct their synthesis. These interactions have involved the hybridization of complementary, or antisense, oligonucleotides or oligonucleotides analogs to mRNA. Hybridization is the sequence-specific hydrogen bonding of an oligonucleotide or oligonucleotide analog to an mRNA sequence via Watson-Crick hydrogen bond formation. Interfering with the production of proteins involved in disease would provide maximum therapeutic results with minimum side effects.
The pharmacological activity of antisense oligonucleotides and oligonucleotide analogs depends on a number of factors that influence the effective concentration of these agents at specific intracellular targets. One important factor for oligonucleotides and analogs thereof is their stability to nucleases. It is unlikely that unmodified oligonucleotides containing phosphodiester linkages will be useful therapeutic agents because they are rapidly degraded by nucleases. Modified oligonucleotides which are nuclease resistant are therefore greatly desired.
Phosphorothioate and phosphorodithioate oligonucleotide analogs which have one or both of the non-bridging oxygens of the natural phosphodiester linkage replaced with sulphur, respectively, are especially promising antisense therapeutics. These oligonucleotide analogs are highly resistant to nucleases, have the same charge as natural phosphodiester-containing oligonucleotides, and are taken up by cells in therapeutically effective amounts. See, for example, Baracchini et al, U.S. Pat. No. 5,510,239; Ecker, U.S. Pat. No. 5,512,438; Bennett et al, U.S. Pat. No. 5,514,788; and Ecker et al, U.S. Pat. No. 5,523,389.
Phosphorothioate and phosphorodithioate oligonucleotide analogs are conveniently synthesized with automated DNA synthesizers using hydrogen phosphonate chemistry which permits the phosphonate backbone to be sulfurized in a single step after automated synthesis. One drawback of this approach is that coupling yields during chain synthesis are typically lower than those obtained using phosphoramidite chemistry. The final yield of the desired oligonucleotide analog is therefore too low due to the low individual coupling yields.
Automated synthesis using phosphoramidite chemistry is a highly desirable approach to the synthesis of these sulfurized oligonucleotide analogs, with coupling yields typically greater than 99%. However, the phosphorous(III)-containing phosphite intermediates are unstable under the conditions of the detritylation step of the synthesis cycle. Therefore, these phosphorous(III) linkages must be sulfurized after each coupling step.
A more recent method for the synthesis of oligonucleotide analogs is the xe2x80x9cblockmerxe2x80x9d approach. In blockmer synthesis, an oligonucleotide analog is made by the sequential coupling of short protected oligomers or blocks, e.g., a dinucleotide, on a solid support. This strategy offers several advantages over the conventional synthetic approach which involves the sequential coupling of monomeric nucleoside phosphoramidites. The number of synthesis cycles required to prepare an oligonucleotide analog is reduced, saving time and minimizing reagent consumption. The blocks may be prepared on a large scale using inexpensive solution phase synthesis techniques. In order to prepare sulfurized oligonucleotide analogs by the blockmer method, a reagent for sulfuring the phosphorous(III) linkages of the blocks on a large scale is required. The blockmer approach is described in the following references: Ravikumar et al, WO 95/32980; WO 94/15947; Journal of Organic Chemistry 1984, 49, 4905-4912; Helevetica Chimica Acta 1985, 68, 1907-1913; Chem. Pharm. Bull. 1987, 35, 833-836.
There are several reagents available for sulfurizing the phosphite intermediates during automated oligonucleotide synthesis. All of these reagents have drawbacks which limit their use for synthesizing sulfurized oligonucleotide analogs.
Elemental sulfur, for example, has been used to sulfurize phosphorous(III) linkages in solid phase oligonucleotide synthesis. However, elemental sulphur is not suitable for use with automated synthesizers because of its poor solubility in standard solvents and slow sulfurization rate. In addition, carbon disulfide, the preferred solvent for elemental sulphur, is highly volatile and has a low flash point. See, U.S. Pat. No. 5,252,723 and U.S. Pat. No. 5,449,769.
The Beaucage reagent, 3H-1,2-benzodithiol-3-one, is a considerably more efficient sulfurizing agent. However, this reagent precipitates from solution and clogs the solvent and reagent transfer lines of an automated DNA synthesizer. Also, the by-product formed during the sulfurization reaction is a potent oxidizing agent. This by-product can lead to side products, e.g., phosphodiesters, which are difficult to separate from the desired sulfurized oligonucleotides. In addition, the preparation of this reagent involves expensive and toxic materials, and is therefore not amenable for large-scale synthesis of sulfurized oligonucleotide analogs. See, U.S. Pat. No. 5,003,097.
Tetraethylthiuram disulfide is an inexpensive and chemically stable sulfurization reagent. However, the sulfurization rate is slow and therefore a significant molar excess of this reagent is required. Even with an excess of this reagent, sulfurization yields are unacceptably low. See, U.S. Pat. No. 5,166,387.
Phenylacetyl disulfide may be used to sulfurize phosphite intermediates during automated oligonucleotide synthesis. However, this reagent has not been reported to be useful for large-scale synthesis of sulfurized oligonucleotide analogs. See, Recherches Travaux Chimiques des Pays-Bas 1991, 110, 325-331; Tetrahedron Letters 1989, 30, 6757-6760; Synthesis 1981, 637-638.
Accordingly, there remains a need in the art for methods and reagents for synthesizing sulfurized oligonucleotide analogs which overcome these problems.
An object of the present invention is to provide a method of synthesizing a sulfurized oligonucleotide using a reagent that efficiently sulfurizes a phosphorous(III) linkage in an oligonucleotide analog.
Another object of the present invention is to provide a method of synthesizing a sulfurized oligonucleotide using a reagent that does not require the use of solvents having a high volatility and flash point.
Another object of the present invention is to provide a method of synthesizing a sulfurized oligonucleotide using a reagent that is highly soluble in organic solvents and does not precipitate from solution.
Another object of the present invention is to provide a method of synthesizing a sulfurized oligonucleotide using a reagent that is useful in automated solid phase synthesis of oligonucleotide analogs.
Another object of the present invention is to provide a method of synthesizing a sulfurized oligonucleotide using a reagent that is compatible with large-scale and small-scale synthesis using solution phase methods.
Another object of the present invention is to provide a sulfurizing reagent composition that may be used to sulfurize a phosphorous(III) linkage of an oligonucleotide analog.
These objects and others may be accomplished with a method of synthesizing a sulfurized oligonucleotide analog by reacting an oligonucleotide analog containing a phosphorus(III) linkage capable of being sulfurized with a thiodicarbonic acid diester polysulfide having the formula: 
where each R is an inert side chain, and n is 2, 3 or 4.
The above objects may also be accomplished with a method of synthesizing a sulfurized oligonucleotide analog by:
(a) providing a nucleoside analog having a blocked hydroxyl group;
(b) deblocking the blocked hydroxyl group to produce a free hydroxyl group;
(c) reacting the free hydroxyl group with a protected nucleoside analog phosphoramidite or a protected nucleoside analog phosphorothioamidite having a blocked hydroxyl group to produce an oligonucleotide analog containing a phosphorous(III) linkage and a blocked hydroxyl group;
(d) reacting the phosphorous(III) linkage with a reagent selected from the group consisting of an oxidizing agent and a dithiocarbonic acid diester polysulfide to produce an oxidized or sulfurized phosphorous(V) linkage;
(e) repeating steps (b) through (d) at least once to produce a sulfurized oligonucleotide analog, wherein at least one step (d) in the method is reacting the phosphorous(III) linkage with the dithiocarbonic acid diester polysulfide of the present invention.
The above objects may also be accomplished with a sulfurizing reagent composition containing an effective amount of thiodicarbonic acid diester polysulfide for sulfurizing a phosphorous(III) linkage of an oligonucleotide analog and at least one solvent.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description.
As used in the present invention, the term xe2x80x9coligonucleotide analogxe2x80x9d includes linear oligomers of natural or modified nucleosides linked by phosphodiester bonds or analogs thereof ranging in size from two monomeric units to several hundred monomeric units. Oligonucleotide analogs include modifications of the heterocyclic base moiety and/or the sugar portion of a component nucleotide. In particular, the term includes non-natural oligomers containing phosphorus(III) linkages which are amenable to sulfurization. Preferably, the modifications do not inhibit the ability of an oligonucelotide analog to bind to a target nucleic acid. The term xe2x80x9csulfurized oligonucleotide analogxe2x80x9d is an oligonucleotide analog containing at least one analog of a phosphodiester linkage in which one or both of the non-bridging oxygen atoms are replaced by sulfur. The term xe2x80x9cnucleoside analogxe2x80x9d refers to a natural or modified nucleoside. In particular, this term includes nucleosides that are modified at the heterocyclic base and/or sugar to enhance hybridization to the target nucleic acids. It is to be understood that the stereochemical relationship between the sugar substituents in the nucleoside and oligonucleotide analogs disclosed herein is preferably the same as that of naturally-occurring DNA and RNA, see G. M. Blackburn and M. J. Gait (eds.), Nucleic Acids in Chemistry and Biology (ILR Press, 1990), Chapter 2, pp. 19-70.
The thiodicarbonic acid diester polysulfide of the present invention preferably has the formula: 
where each R is preferably an inert group. These groups preferably do not contain any reactive moieties which could lead to side reactions or poor yields in the sulfurization reaction. Preferably, each R is independently C1-C8 alkyl, substituted C1-C8 alkyl, C2-C8 heterocycloalkyl containing up to three heteroatoms, substituted C2-C8 heterocycloalkyl containing up to three heteroatoms, C6-C14 aryl, substituted C6-C14 aryl, C3-C11 hetaryl containing up to three heteroatoms, substituted C3-C11 hetaryl containing up to three heteroatoms, C7-C18 aralkyl, substituted C7-C18 aralkyl, C4-C15 heterocycloaralkyl containing up to three heteroatoms or substituted C4-C15 heterocycloaralkyl containing up to three heteroatoms. The term xe2x80x9cC1-C8 alkylxe2x80x9d includes linear, branched and cyclic alkyl groups. The term xe2x80x9csubstitutedxe2x80x9d means that up to three hydrogen atoms in the group are substituted with up to three halogen, nitro, cyano, C1-C8 alkyl, Oxe2x80x94C1-C8 alkyl, Nxe2x80x94C1-C8 alkyl, Sxe2x80x94C1-C8 alkyl groups or combinations thereof. More preferably, each R is independently C2-C8 alkyl, C6-C14 aryl or substituted C6-C14 aryl. Most preferably, each R is independently ethyl or p-chlorophenyl. The dithiocarbonic acid diester polysulfide may be a disulfide, trisulfide or tetrasulfide, i.e., n is 2, 3 or 4, respectively. Preferably n is 2 or 3, and more preferably, n is 2.
The thiodicarbonic acid diester polysulfide may be prepared by oxidation of the corresponding thiodicarbonic acid or acid salt with oxidizing agents, such as iodine or bromine. Methods of synthesis and properties of these polysulfides are described in the following references: Barany et al, Journal of Organic Chemistry, 1983, 48, 4750-4761; W. F. Zeise, J. Prakt. Chem. 1845, 36, 352-362; Losse et al., J. Prakt. Chem. 1961, 13, 260; S. R. Rao, Xanthates and Related Compounds, (M. Decker, New York, 1971); Bulmer et al, J. Chem. Soc. 1945, 674-677.
The thiodicarbonic acid diester polysulfide of the present invention is an efficient reagent for sulfurizing phosphorous(III) linkages in oligonucleotide analogs. The reagent does not precipitate out of solution, even on prolonged storage. The reagent may be used in solution phase synthesis and is particularly useful in solid-phase synthesis of oligonucleotide analogs with automated DNA synthesizers. The reagent is particularly useful in large-scale synthesis, using either solution phase or solid phase techniques.
When used to sulfurize phosphorus(III) linkages in oligonucleotide analogs, the thiodicarbonic acid diester polysulfide is preferably delivered to the oligonucleotide analog in a suitable organic solvent, such as acetonitrile, pyridine, tetrahydrofuran, dichloromethane, dichloroethane and collidine. These solvents may be used singly or as mixtures in any proportion. Preferable solvents are pyridine, dichloromethane and mixtures thereof. Pyridine is most preferred. The reagent may be used at any effective concentration for sulfurizing a phosphorous(III) linkage, preferably between 0.01 M to 1.5 M, more preferably from 0.2 to 1.2 M; and most preferably from 0.5 to 1.0 M.
The sulfurization reaction may be conducted at any convenient temperature, preferably from 0 to 70xc2x0 C.; more preferably from 10 to 40xc2x0 C.; and most preferably at about room temperature, i.e., 18 to 25xc2x0 C. The sulfurization reaction is preferably conducted for 30 seconds to 15 minutes, more preferably, 1 to 15 minutes; and most preferably, 3 to 10 minutes. Preferably, sulfurization is performed under anhydrous conditions with the exclusion of air.
The present method for synthesizing a sulfurized oligonucleotide analog may be applied to any oligonucleotide analog containing at least one phosphorus(III) linkage which is amenable to sulfurization. In particular, the present method is useful for sulphurizing phosphite triesters, thiophosphite triesters, and hydrogen phosphonates. More preferably, the phosphorus(III) linkage is a phosphite triester or a thiophosphite triester. Most preferably, the phosphorus(III) linkage is a phosphite triester.
Detailed procedures for synthesizing oligonucleotide analogs containing at least one phosphorus(III) linkages are well-known in the art and are described in the following references: M. J. Gait (ed.), Oligonucleotide Synthesis, A Practical Approach (ILR Press, 1984); J. S. Cohen (ed.), Oligonucleotides: Antisense Inhibitors of Gene Expression, (CRC Press, Inc., Boca Raton, Fla., 1989).
Preferably, the dithiocarbonic acid diester polysulfide of the present invention is used in conjunction with the phosphoramidite or phosphorothioamidite synthetic approaches. Synthesis may be conducted in solution phase or using solid phase techniques. More preferably, the synthesis is conducted using a solid support. Most preferably, the synthesis is conducted on a solid support using an automated DNA synthesizer, e.g., an APPLIED BIOSYSTEMS model 380B or a similar machine.
Preferably, this synthetic approach involves the following steps: (1) deprotecting a blocked reactive functionality on the growing oligonucleotide analog chain or on the first nucleoside analog monomer, to produce a deblocked reactive functionality, (2) reacting an appropriately blocked and protected nucleoside analog phosphoramidite or phosphorothioamidite monomer with the deblocked reactive functionality of the growing nucleotide analog chain, preferably in the presence of an activator, to form an oligonucleotide analog containing a phosphorus(III) linkage, (3) capping any unreacted functionalities, and (4) sulfurizing the newly-formed phosphorus(III) linkage with the thiodicarbonic acid diester polysulfide to obtain the phosphorus atom in a sulfurized pentacoordinate state.
The term xe2x80x9cblockedxe2x80x9d means that a reactive functionality, usually a nucleophile, e.g., a 5xe2x80x2 hydroxyl, is protected with a group that may be selectively removed. Preferably, these blocking groups are labile to dilute acid, e.g. dichloroacetic acid in dichloromethane, and are stable to base. Preferable blocking groups include 4,4xe2x80x2-dimethoxytrityl (DmTr), monomethoxytrityl, diphenylmethyl, phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthen-9-yl (Mox). The 4,4xe2x80x2-dimethoxytrityl group is most preferred. Throughout the present disclosure the labile 5xe2x80x2 blocking group is represented as xe2x80x9cR6xe2x80x9d.
Any natural or non-natural heterocyclic base may be used in the present invention, such as adenine, guanine, cytosine, thymine, uracil, 2-aminopurine, inosine, substituted pyrimidines, e.g., 5-methylcytosine, and 5-nitropyrrole. Other suitable heterocyclic bases are described by Merigan et al., U.S. Pat. No. 3,687,808. Preferably, the heterocyclic base is attached to C-1 of the sugar moiety of nucleoside analog phosphoramidite (1) via a nitrogen of the base. Throughout the present disclosure the heterocyclic base is respresented as xe2x80x9cBxe2x80x9d.
During synthesis these heterocyclic groups are preferably protected to prevent any reactive group, e.g., an exocyclic amino group, to prevent undesired side reactions. The term xe2x80x9cprotectedxe2x80x9d means that reactive moieties such as exocyclic amino groups, 2xe2x80x2-hydroxyl groups, oxygen or sulfur bonded to phosphorus atoms, and the like, have protective groups which are generally removed after synthesis of the oligonucleotide analog is completed. Preferably, these protective groups are labile to a base and/or a nucleophile. This term also includes oligonucleotide and nucleoside analogs which have groups that do not require such protection, e.g., heterocyclic bases such as thymine or abasic nucleosides.
Preferable protecting groups for the heterocyclic bases include base labile groups. The exocyclic amino groups of the heterocyclic groups are preferably protected with acyl groups that are removed by base treatment after synthesis of the sulfurized oligonucleotide analog. Preferably, these protecting groups are C2-C10 acyl groups. N-benzoyl and N-isobutyryl protecting groups are particularly preferred. Adenine is preferably protected as an N2-isobutyryl derivative. Guanine is preferably protected as an N6-isobutyryl derivative. Cytidine is preferably protected as an N4-benzoyl derivative.
The sulfurized nucleotide analogs of the present invention may be substituted at the 2xe2x80x2 position. Preferable 2xe2x80x2 substituents are groups that enhance the hybridization of an oligonucleotide analog with its target nucleic acid, a group that improves the in vivo stability of an oligonucleotide analog or enhances the pharmacokinetic and/or pharamacodynamic properties of an oligonucleotide analog.
Examples of 2xe2x80x2 substituents include hydrogen, hydroxyl, F, Cl, Br, C1-C8 alkyl, substituted C1-C8 alkyl, C2-C8 heterocycloalkyl containing up to three heteroatoms, substituted C2-C8 heterocycloalkyl containing up to three heteroatoms, C6-C14 aryl, substituted C6-C14 aryl, C3-C11 hetaryl containing up to three heteroatoms, substituted C3-C11 hetaryl containing up to three heteroatoms, C7-C18 aralkyl, substituted C7-C18 aralkyl, C4-C15 heterocycloaralkyl containing up to three heteroatoms, substituted C4-C15 heterocycloaralkyl containing up to three heteroatoms, Oxe2x80x94C1-C8 alkyl, substituted Oxe2x80x94C1-C8 alkyl (such as CF3), Oxe2x80x94C2-C8 heterocycloalkyl containing up to three heteroatoms, substituted Oxe2x80x94C2-C8 heterocycloalkyl containing up to three heteroatoms, Oxe2x80x94C6-C14 aryl (such as phenyl), substituted Oxe2x80x94C6-C14 aryl, Oxe2x80x94C3-C11 hetaryl containing up to three heteroatoms, substituted Oxe2x80x94C3-C11 hetaryl containing up to three heteroatoms, Oxe2x80x94C7-C18 aralkyl (such as benzyl), substituted Oxe2x80x94C7-C18 aralkyl, Oxe2x80x94C4-C15 heterocycloaralkyl containing up to three heteroatoms, substituted Oxe2x80x94C4-C15 heterocycloaralkyl containing up to three heteroatoms, Oxe2x80x94C1-C8-alkyl-Oxe2x80x94C1-C8-alkyl, Oxe2x80x94C1-C8 alkenyl, Oxe2x80x94C1-C8 alkoxyamino, O-tri-C1-C8-alkyl silyl (such as tert-butyldimethylsilyl), substituted O-tri-C1-C8-alkyl silyl, NHxe2x80x94C1-C8 alkyl, Nxe2x80x94(C1-C8)2, NHxe2x80x94C1-C8 alkenyl, Nxe2x80x94(C1-C8)2 alkenyl, Sxe2x80x94C1-C8 alkyl, Sxe2x80x94C1-C8 alkenyl, NH2, N3, NHxe2x80x94C1-C8-alkyl-NH2, polyalkylamino and an RNA cleaving group. Preferable RNA cleaving groups include the O-{3-propoxy-[2-naphthyl-7-(1-(dimethylaminosulfonyl)-imidazol-4-yl)]} group and the O-{3-propoxy-[2-naphthyl-7-(1-(dimethylaminosulfonyl-2-methoxy-5-acetylaminomethyl)-imidazol-4-yl)]} group. These groups are discussed by Cook et al, U.S. Pat. No. 5,359,051.
Preferably, the 2xe2x80x2 substituents are hydrogen, hydroxyl, Oxe2x80x94C1-C8 alkyl, F, Oxe2x80x94C1-C8-alkoxyamino and Oxe2x80x94C1-C8-alkyl-Oxe2x80x94C3-C8-alkyl. More preferably, the 2xe2x80x2 substituents are hydrogen, Oxe2x80x94C1-C8 alkyl, F and Oxe2x80x94C1-C8-alkyl-Oxe2x80x94C1-C8-alkyl. Most preferably, the 2xe2x80x2 substituent is hydrogen or a methoxyethoxy group. Throughout the present disclose, the 2xe2x80x2 substituent is respresented as xe2x80x9cXxe2x80x9d.
A variety of protecting groups for the oxygen and sulfur atoms attached to the phosphorus atom in the nucleoside analog phosphoramidite and phosphorothioamidite, respectively, may be used. These protecting groups are preferably removed at after synthesis is complete. Preferably, these protecting groups are labile to a base and/or a nucleophile. Most prefereably, these protecting groups are removed by aqueous ammonium hydroxide. Preferable protecting groups are 2-cyanoethyl, 4-cyano-2-butenyl, 2-diphenylmethylsilylethyl (DPSE) or a 2-N-amidoethyl group having the formula R1CONR2CHR3CHR4xe2x80x94. These protecting groups are preferably removed after synthesis, preferably with an aqueous solution of ammonia at a temperature between room temperature and 75xc2x0 C. In the present invention, the terms xe2x80x9cphosphodiester linkagexe2x80x9d, xe2x80x9cphosphorothioate linkagexe2x80x9d and xe2x80x9cphosphorodithioate linkagexe2x80x9d describe these internucleosidic linkages in protected or unprotected form. Throughout the present disclosure, these phosphorous protecting groups will be represented as xe2x80x9cPgxe2x80x9d. An oxygen atom or sulfur atom attached to the phosphorous atom in a nucleoside analog phosphoramidite or thiophosphoramidite or an oligonucleotide analog is represenated as xe2x80x9cYxe2x80x9d. Preferably, Y is an oxygen atom.
The 4-cyano-2-butenyl protecting group is removed by xcex4-elimination, preferably using the standard NH3/H2O deprotection conditions known in the art. 4-cyano-2-butenyl-protected nucleoside analog phosphoramidites may be prepared with 4-cyano-2-butene-1-ol and appropriately protected nucleoside analogs using known synthetic methodology. The synthesis of 4-cyano-2-butene-1-ol is disclosed by Ravikumar et al, Synthetic Communications 1996, 26(9), 1815-1819.
The 2-diphenylmethylsilylethyl (DPSE) protecting group is described in Ravikumar et al., WO 95/04065. This protecting group may be removed by treatment with a base, preferably aqueous ammonium hydroxide. The DPSE group may also be removed with fluoride ion. Preferably, the fluoride ion is provided from a salt such as a tetraalkylammonium fluoride, e.g., tetrabutylammonium fluoride (TBAF) or an inorganic fluoride salt, e.g., potassium fluoride or cesium fluoride in a solvent such as tetrahydrofuran, acetonitrile, dimethoxyethane or water.
The 2-N-amidoethyl group is described in the commonly assigned application U.S. patent application Ser. No. 08/811,232 (Title: Protecting Group for Synthesizing Oligonucleotide Analogs, Attorney Docket No. 7761-002-55). The 2-N-amidoethyl group has the formula R1CONR2CHR3CHR4xe2x80x94, where R1 is C1-C8 alkyl, substituted C1-C8 alkyl, C2-C8 heterocycloalkyl containing up to three heteroatoms, substituted C2-C8 heterocycloalkyl containing up to three heteroatoms, C6-C14 aryl, substituted C6-C14 aryl, C3-C11 hetaryl containing up to three heteroatoms, substituted C3-C11 hetaryl containing up to three heteroatoms, C7-C18 aralkyl, substituted C7-C18 aralkyl, C4-C15 heterocycloaralkyl containing up to three heteroatoms or substituted C4-C15 heterocycloaralkyl containing up to three heteroatoms. More preferably, R1 is C1-C8 alkyl, substituted C1-C8 alkyl, C2-C8 heterocycloalkyl containing up to three heteroatoms, substituted C2-C8 heterocycloalkyl containing up to three heteroatoms, C6-C14 aryl, substituted C6-C14 aryl, C3-C11 hetaryl containing up to three heteroatoms or substituted C3-C11 hetaryl containing up to three heteroatoms. Even more preferably, R1 is methyl, fluoromethyl, difluoromethyl or trifluoromethyl. Most preferably, R1 is methyl, trifluoromethyl or phenyl.
The nitrogen atom of the 2-N-amidoethyl group may be unsubstituted, i.e., R2 may be hydrogen, or substituted. Preferably, R2 is hydrogen, C1-C8 alkyl, substituted C1-C8 alkyl, C2-C8 heterocycloalkyl containing up to three heteroatoms, substituted C2-C8 heterocycloalkyl containing up to three heteroatoms, C6-C14 aryl, substituted C6-C14 aryl, C3-C11 hetaryl containing up to three heteroatoms, substituted C3-C11 hetaryl containing up to three heteroatoms, C7-C18 aralkyl, substituted C7-C18 aralkyl, C4-C15 heterocycloaralkyl containing up to three heteroatoms or substituted C4-C15 heterocycloaralkyl containing up to three heteroatoms. More preferably, R2 is hydrogen or C1-C8 alkyl. Most preferably, R2 is hydrogen or methyl.
The ethyl moiety of the 2-N-amidoethyl group may be unsubstituted, e.g., R3 and R4 may both be hydrogen. Alternatively, the ethyl moiety may be substituted with groups that preferably do not compromise the stability of the 2-N-amidoethyl group during oligonucleotide analog synthesis and permit the protecting group to be removed by treatment with a base and/or a nucleophile following step-wise assembly of an oligonucleotide analog. Preferable R3 and R4 groups are hydrogen, C1-C8 alkyl, substituted C1-C8 alkyl, C2-C8 heterocycloalkyl containing up to three heteroatoms, substituted C2-C8 heterocycloalkyl containing up to three heteroatoms, C6-C14 aryl, substituted C6-C14 aryl, C3-C11 hetaryl containing up to three heteroatoms, substituted C3-C11 hetaryl containing up to three heteroatoms, C7-C18 aralkyl, substituted C7-C18 aralkyl, C4-C15 heterocycloaralkyl containing up to three heteroatoms or substituted C4-C15 heterocycloaralkyl containing up to three heteroatoms. More preferably, R3 is hydrogen or linear C1-C8 alkyl. Most preferably, R3 is hydrogen or methyl. More preferably, R4 is hydrogen, C6-C14 aryl, substituted C6-C14 aryl, C3-C11 hetaryl containing up to three heteroatoms or substituted C3-C11 hetaryl containing up to three heteroatoms. Most preferably, R4 is hydrogen or phenyl. The R3 and R4 groups are independently selected, i.e., they may be the same or different.
Alternatively, R3 and R4, together with the carbon atoms they are bonded to, may form a C3-C8 cycloalkyl group, a substituted C3-C8 cycloalkyl group, a C2-C8 heterocycloalkyl group containing up to three heteroatoms or a substituted C2-C8 heterocycloalkyl group containing up to three heteroatoms. In this embodiment, R3 and R4, together with the carbon atoms they are bonded to, preferably form a C3-C8 cycloalkyl group or a substituted C3-C8 cycloalkyl group. More preferred cycloalkyl groups are C4-C7 or substituted C4-C7 groups, with C5-C6 or substituted C5-C6 groups most preferred. An unsubstituted cycloalkyl group is particularly preferred. An unsubstituted C6 cycloalkyl group is most particularly preferred. The stereochemical relationship between the N-amido group and Y may be cis or trans. A trans relationship is preferred.
An allyl group is also a preferable protecting group for the oxygen or sulfur atom attached to the phosphorus atom in an oligonucleotide analog. The allyl protecting group is described in U.S. Pat. No. 5,026,838. The term xe2x80x9callyl groupxe2x80x9d includes allyl, methallyl, crotyl, prenyl, geranyl, cinnamyl and p-chlorocinnamyl groups. The number of carbon atoms in these groups is preferably 3 to 10. Preferably, the allyl group is an unsubstituted allyl group. The allyl group may be removed with a palladium(0) compound and a nucleophilic agent, such as an amine or a formic acid salt, under neutral conditions at room temperature. A preferred reagent is tetrakis(triphenylphosphine) palladium(0) and n-butylamine in tetrahydrofuran.
An activator is generally used in the coupling of a deblocked reactive functionality on the oligonucleotide or nucleoside analog and the phosphoramidite or phosphorothioamidite monomer. Preferable activators are well-known in the art, such as 1H-tetrazole, 5-(4-nitrophenyl)-1H-tetrazole and diisopropylamino tetrazolide. 1H-tetrazole is most preferred.
A capping step is preferably used after this coupling reaction to permanently block all uncoupled reactive functionalities. Suitable capping reagents are well-known in the art. A preferable capping reagent is acetic anhydride/lutidine/THF (1:1:8) with N-methylimidazole/THF.
When synthesis is performed by solution phase methods, the 3xe2x80x2 terminal hydroxyl group of an oligonucleotide analog is preferably protected to prevent the 3xe2x80x2 hydroxyl group from participating in any undesired side reactions. Preferably, the terminal 3xe2x80x2 hydroxyl group is protected with a group which may be removed selectively without removing any other protecting groups. A C2-C10 acyl group is preferred. The acetyl or levulinyl group is more preferred. The levulinyl group is most preferred. Throughout the present disclosure this 3xe2x80x2 protecting group is represented as xe2x80x9cR7xe2x80x9d.
The sequential addition of nucleoside analog phosphoramidites or phosphorothioamidites may be repeated until an oligonucleotide analog having the desired sequence length is obtained. The length of the sulfurized oligonucleotide analog is preferably 2 to 200 monomer units; more preferably, 2 to 100 monomer units; even more preferably, 2 to 50 monomer units; and, most preferably, 2 to 25 monomer units. These ranges include all subranges therebetween.
In a preferred embodiment of the present invention, a sulfurized oligonucleotide analog is synthesized on a solid support. Suitable solid supports include controlled pore glass (CPG); oxalyl-controlled pore glass, see for example Alul et al, Nucleic Acids Research 1991, 19,xe2x80x941527; TENTAGEL Support, see Wright et al, Tetrahedron Letters 1993, 34, 3373; POROS, a polystyrene resin available from PERCEPTIVE BIOSYSTEMS; and a polystyrene/divinylbenzene copolymer. Controlled pore glass is the most preferred solid support. Throughout the present discloure the solid support is represented as xe2x80x9cSpxe2x80x9d.
The oligonucleotide analog is preferably attached to the it solid support by a group which may be easily cleaved to release the oligonucleotide analog from the solid support when synthesis is complete. Preferably, this group may be cleaved upon exposure to a base and/or a nucleophile. More preferably, the group is an acyl. Most preferably, the group is a carboxyl group esterifed with the terminal 3xe2x80x2 hydroxyl group of the oligonucleotide analog. The group linking an oligonucleotide analog to a solid support is represented as xe2x80x9cLxe2x80x9d in the present disclosure.
The present invention includes sulfurized oligonucleotide analogs containing phosphorothioate, phosphorodithioate, and phosphodiester linkages in any combination. The sulfurized oligonucleotide analogs of the present invention may contain only sulfurized linkages, e.g., phosphorothioate and/or phosphorodithioate. The oligonucleotide analogs may also contain one or more phosphodiester linkages in addition to the sulfurized linkages. In a preferred embodiment, the sulfurized oligonucleotide analog contains both phosphorothioate and phosphodiester linkages. In another preferred embodiment, the oligonucleotide contains both phosphorodithioate and phosphodiester linkages.
Phosphodiester linkages are formed by oxidizing a phosphorous(III) linkage with any suitable oxidizing reagent known in the art, e.g., I2/THF/H2O, H2O2/H2O, tert-butyl hydroperoxide or a peracid, such as m-chloroperbenzoic acid. I2/THF/H2O is a preferred oxidizing agent.
In a preferred embodiment, the oligonucleotide analog containing at least one phosphorous(III) linkage has the formula: 
where
each Pg is independently a group labile to a base and/or a nucleophile or an allyl group;
R6 is a labile blocking group;
each B is independently an unprotected or protected heterocyclic base;
each X is independently selected from the group consisting of hydrogen, hydroxyl, F, Cl, Br, C1-C8 alkyl, substituted C1-C8 alkyl, C2-C8 heterocycloalkyl containing up to three heteroatoms, substituted C2-C8 heterocycloalkyl containing up to three heteroatoms, C6-C14 aryl, substituted C6-C14 aryl, C3-C11 hetaryl containing up to three heteroatoms, substituted C3-C11 hetaryl containing up to three heteroatoms, C7-C18 aralkyl, substituted C7-C18 aralkyl, C4-C15 heterocycloaralkyl containing up to three heteroatoms, substituted C4-C15 heterocycloaralkyl containing up to three heteroatoms, Oxe2x80x94C1-C8 alkyl, substituted Oxe2x80x94C1-C8 alkyl, Oxe2x80x94C2-C8 heterocycloalkyl containing up to three heteroatoms, substituted Oxe2x80x94C2-C8 heterocycloalkyl containing up to three heteroatoms, Oxe2x80x94C6-C14 aryl, substituted Oxe2x80x94C6-C14 aryl, Oxe2x80x94C3-C11 hetaryl containing up to three heteroatoms, substituted Oxe2x80x94C3-C11 hetaryl containing up to three heteroatoms, Oxe2x80x94C7-C18 aralkyl, substituted Oxe2x80x94C7-C18 aralkyl, Oxe2x80x94C4-C15 heterocycloaralkyl containing up to three heteroatoms, substituted Oxe2x80x94C4-C15 heterocycloaralkyl containing up to three heteroatoms, Oxe2x80x94C1-C8-alkyl-Oxe2x80x94C1-C8-alkyl, Oxe2x80x94C1-C8 alkenyl, Oxe2x80x94C1-C8 alkoxyamino, O-tri-C1-C8-alkyl silyl, substituted O-tri-C1-C8-alkyl silyl, NHxe2x80x94C1-C8 alkyl, Nxe2x80x94(C1-C8)2, NHxe2x80x94C1-C8 alkenyl, Nxe2x80x94(C1-C8)2 alkenyl, Sxe2x80x94C1-C8 alkyl, Sxe2x80x94C1-C8 alkenyl, NH2, N3, NHxe2x80x94C1-C8-alkyl-NH2, polyalkylamino and an RNA cleaving group;
each Y is independently O or S;
each Z is independently O or S;
L is a group labile to a nucleophile and/or base;
Sp is a solid support; and
m is 0 or a positive integer.
Preferred protecting groups, Pg, include 2-cyanoethyl, 4-cyano-2-butenyl, 2-diphenylmethylsilylethyl (DPSE) and a 2-N-amidoethyl group.
In a preferred embodiment, the oligonucleotide analog containing a phosphorous(III) linkage contains 2 to 100 monomer units, i.e., m is 0 to 98; more preferably, from 2 to 50 monomer units, i.e., m is 0 to 48; and most preferably from 2 to 25 monomer units, i.e., m is 0 to 23.
Treating the above oligonucleotide analog with a dithiocarbonic acid diester polysulfide affords the corresponding sulfurized oligonucleotide analog having the formula: 
where Pg, R6, B, X, Y, Z, L, S and m are defined above. Z is an oxygen atom or a sulfur atom. Appropriate choice of Z allows the internucleosidic linkage containing Z to be a phosphodiester, phosphorothioate or phosphorodithioate linkage, depending on selection of Y. For example, when Y and Z are both oxygen the linkage is a phosphodiester. When Y is oxygen and Z is sulfur, or vice versa, the linkage is a phosphorothioate. When V and Z are both sulfur the linkage is a phosphorodithioate. Preferably, Y is an oxygen atom and Z is a sulfur atom which has been introduced using the dithiocarbonic acid diester polysulfide described above. It is to understood that the terms phosphodiester, phosphorothioate and phosphorodithioate refer to the internucleotide linkage after removal of the Pg groups.
Following sulfurization, the sequence of the sulfurized oligonucleotide analog may be further extended. Alternatively, the sulfurized oligonucleotide analog may be deprotected and removed from the solid support to afford the corresponding deprotected sulfurized analog. The deprotected sulfurized analog may contain phosphorothioate, phosphorodithioate and phosphodiester linkages, in any combination.
In another preferred embodiment of the present invention, the oligonucleotide analog containing at least one phosphorous(III) linkage is a dinucleotide having the formula: 
where R7 is a group labile to a base and/or a nucleophile. Sulfurization of the dinucleotide analog with the dithiocarbonic acid diester polysulfide affords the corresponding sulfurized dinucleotide analog having the formula: 
The present invention also provides a method for synthesizing a sulfurized oligonucleotide analog by:
(a) providing a nucleoside analog having a blocked hydroxyl group;
(b) deblocking the blocked hydroxyl group to obtain a free hydroxyl group;
(c) reacting the free hydroxyl group with a protected nucleoside analog phosphoramidite having a blocked hydroxyl group to produce an oligonucleotide analog containing a phosphorous(III) linkage and a blocked hydroxyl group;
(d) reacting the phosphorous(III) linkage with a reagent selected from the group consisting of an oxidizing agent and a dithiocarbonic acid diester polysulfide having the formula: 
xe2x80x83to produce an oxidized or sulfurized phosphorous(V) linkage;
(e) repeating steps (b) through (d) at least once to produce a sulfurized oligonucleotide analog; where the oligonucleotide analog contains at least one sulfurized phosphorous(V) linkage. Preferably, each phosphorous(V) linkage is sulfurized.
A preferred nucleoside analog phosphoramidite having a blocked hydroxyl group used in step (c) has the formula: 
The R5 groups are chosen such that the nucleoside analog phosphoramidite preferably couples efficiently with a reactive group on the on the growing oligonucleotide analog chain, e.g., a 5xe2x80x2 hydroxyl group, to form a phosphorous(III) internucleotide linkage. The R5 groups are independently selected, i.e., they may be the same or different. Preferable R5 groups are C1-C8 alkyl, substituted C1-C8 alkyl, C2-C8 heterocycloalkyl containing up to three heteroatoms, substituted C2-C8 heterocycloalkyl containing up to three heteroatoms, C6-C14 aryl, substituted C6-C14 aryl, C3-C11 hetaryl containing up to three heteroatoms, substituted C3-C11 hetaryl containing up to three heteroatoms, C7-C18 aralkyl, substituted C7-C18 aralkyl, C4-C15 heterocycloaralkyl containing up to three heteroatoms or substituted C4-C15 heterocycloaralkyl containing up to three heteroatoms; or both R5 groups together with the nitrogen atom they are bonded to form a C2-C8 heterocycloalkyl group containing up to three heteroatoms, a substituted a C2-C8 heterocycloalkyl group containing up to three heteroatoms, a C3-C11 hetaryl group containing up to three heteroatoms or a substituted C3-C11 hetaryl group containing up to three heteroatoms. More preferably, the R5 groups are each a C1-C8 alkyl group or together with the nitrogen atom they are bonded to form a C2-C8 heterocycloalkyl group containing up to three heteroatoms. Even more preferably, each, R5 group is a branched C1-C8 alkyl group. Most preferably, both R5 groups are isopropyl.
In a preferred embodiment, the nucleoside analog in step (a) is attached to a solid support. A preferred nucleoside analog attached to a solid support has the formula: 
In another embodiment, the oligonucleotide analog containing a phosphorous(III) linkage and a blocked hydroxyl group is attached to a solid support and has the formula: 
and the resulting sulfurized oligonucleotide analog has the formula: 
The sulfurized oligonucleotide analog synthesized on a solid support may be removed from the support, preferably by base treatment. Preferably, all of the protecting groups are removed during cleavage from the solid support. Preferable reagents for cleaving the sulfurized oligonucleotide analog from the solid support are aqueous ammonium hydroxide and ammonia/methanol solutions. The simultaneous deprotection and removal of the sulfurized oligonucleotide analog is preferably accomplished in aqueous ammonium hydroxide at a temperature between room temperature, i.e., 18 to 25xc2x0 C., and 75xc2x0 C.; more preferably, between room temperature and 65xc2x0 C.; and most preferably, between room temperature and 60xc2x0 C. A temperature of 55xc2x0 C. is particularly preferred. The deprotection reaction time is preferably 1 to 30 hours; more preferably, 1 to 24 hours; and most preferably, 12-24 hours. The concentration of ammonium hydroxide in the solution used for deprotection is preferably 20 to 30% by weight; more preferably, 25 to 30% by weight; and most preferably, 28 to 30% by weight.
In a preferred embodiment, the sulfurized oligonucleotide analog released from the solid support has the formula: 
where each B is preferably an unprotecteted heterocyclic base.
Each internucleotide linkage of the sulfurized oligonucleotide analog released from the solid support may be ionized, depending on the pH, temperature and salt conditions. Each internucleotide linkage will be ionized in aqueous solution at physiologic pH, temperature and salt conditions, i.e., pH 7.2, 37xc2x0 C. and about 150 mM monovalent salts.